A smart coating with integrated physical antimicrobial and strain-mapping functionalities for orthopedic implants

The prevalence of orthopedic implants is increasing with an aging population. These patients are vulnerable to risks from periprosthetic infections and instrument failures. Here, we present a dual-functional smart polymer foil coating compatible with commercial orthopedic implants to address both septic and aseptic failures. Its outer surface features optimum bioinspired mechano-bactericidal nanostructures, capable of killing a wide spectrum of attached pathogens through a physical process to reduce the risk of bacterial infection, without directly releasing any chemicals or harming mammalian cells. On its inner surface in contact with the implant, an array of strain gauges with multiplexing transistors, built on single-crystalline silicon nanomembranes, is incorporated to map the strain experienced by the implant with high sensitivity and spatial resolution, providing information about bone-implant biomechanics for early diagnosis to minimize the probability of catastrophic instrument failures. Their multimodal functionalities, performance, biocompatibility, and stability are authenticated in sheep posterolateral fusion model and rodent implant infection model.

There are several mechanisms proposed regarding the mechano-bactericidal effect. Some biophysical models suggest that the nanopillar arrays stretch the region of the cell membrane suspended between neighboring nanopillars beyond its elastic limit, resulting in membrane rupture and bacterial death (29). The biomechanical models, one the other hand, indicate that the largest strain, driven by intermolecular adhesion forces, develops at the pillar apex instead of the suspended regions, leading to localized rupture and penetration of the cell envelope (32). Meanwhile, recent proteomic analysis revealed that nanopillars induce oxidative stress within bacterial cells as evident from the elevated levels of oxidative-stress proteins and H2O2 (34).
Here we performed cross-sectional FIB-SEM analysis of the bacteria sitting on top of the bactericidal polymer nanopillar arrays (40,56,57). The bacteria were first incubated on the biomimetic antimicrobial nanopillar arrays (240 nm pitch, 400 nm height, and 100 nm diameter) for 48 hours. After fixation, the areas with bacterial attachment were first identified in top-view SEM, then iteratively cross-sectioned in FIB, and simultaneously imaged via SEM. A 200 nmthick protective layer of platinum was blanketly deposited on top by sputtering before slicing to minimize the charging effect. The cross-sectional FIB-SEM analysis of a S. aureus sitting on top of the polyimide nanopillar arrays was displayed in Fig. S1A and S1B. There were at least four nanopillars in contact with this specific cell (marked as nanopillar #1-4). Three of them (nanopillar #1, 3, and 4) interacted with the side of the S. aureus, only causing the deformation of the bacterial envelope morphology through indentation. The other nanopillar (nanopillar #2), on the contrary, had clearly penetrated the bacterial envelope, but without causing the loss of the turgor pressure. For P. aeruginosa ( Fig. S1C and S1D), at least five neighboring nanopillars were in contact with the cell. The deflection of the nanopillars with bending toward the center of the cell and the penetration through the cell envelope by all nanopillars were both observed. The cell envelope was significantly deformed likely caused by cytoplasmic leakage. The higher probability for nanopillars to pierce through the envelope of Gram-negative bacteria (P. aeruginosa) than that of their Gram-positive counterparts (S. aureus) which are strengthened by a much thicker peptidoglycan cell wall potentially explains the observed quantitative difference in achieved bactericidal efficacy.
The bacterium-nanopillar interface visualized by FIB-SEM shows that the nanopillars are able to pierce through the bacterial envelope and disrupt the continuous barrier between the cytosol and the extracellular environment, which can cause substantial alterations to the bacterial morphology and even the loss of the turgor pressure. These results suggest that the local penetration of the cell membrane at the pillar apex as the plausible mechanism responsible for the mechanobactericidal effect of polymer nanopillar arrays. But further investigations are required to confirm, which will be the subject in our future studies.

Supplementary Text 2: Capillary bending and elastic restoring forces of nanopillars.
High-aspect-ratio nanopillar arrays are susceptible to deformation. Especially, as water evaporates off the surface, the nanopillars bend and could potentially cluster together due to lateral capillary menisci interaction forces (41). Such capillary force Fc between two neighboring nanopillars partially immersed in a liquid can be calculated as 22 22 cos where γ is the liquid-vapor interfacial energy; θ is the equilibrium contact angle; d is the nanopillar diameter; and p is the nanopillar pitch. While the capillary force bends the tips of nanopillars toward each other to form clusters, it is counter-balanced with the elastic restoring force Fe: where E is the modulus, δ is the deflection, and h is the pillar height (58). The clustering can be avoided as long as that Fe > Fc can be maintained over a reasonable range of deflection δ.
By reducing the nanopillar height h by half from 800 nm to 400 nm while keeping both d and p identical, the elastic restoring force Fe is therefore increased by eight times to significantly improve the structural robustness of nanopillars against deformation as shown in Fig. 3F. On the contrary, by reducing the diameter d by half from 100 nm to 50 nm while keeping both p=240 nm and h identical, although the capillary bending force Fc is reduced approximately by half, the elastic restoring force Fe is drastically reduced by 16 times, leading to the enhanced clustering behavior observed in experiment (Fig. 3G).

Supplementary Text 3: Flexural strain on the spinal rod in four-point-bending test.
Fig. S14 illustrates the spinal rod subject to a four-point-bending test. The diameter (d) of the rod (ArteMedics, Grade 23 Titanium) was 5.5 mm. It was attached to two 9 mm-wide steel cubes with screws to limit the rotation of the rod during measurement, which were placed on two supports that were 60 mm apart (Ltotal). The cylindrical actuators (diameter 10 mm) applied two equal point loads up to 2 kN in total on the spinal rod. The distance (L) between the support and the actuator on the same side was 9.5 mm. With the symmetric geometry of the test step, the fore (F) applied at each contact was 1 kN. The maximum deflection recorded in the center of the rod (δ) was ~0.5 mm.
Based on the classical Euler-Bernoulli beam theory, the maximum stress on the rod (σ) can then be calculated as: where M is the moment applied at the middle of the rod defined as: and y is the distance from the neutral plane of the rod to its utmost fibre, which is the rod radius (d/2). 22

(3 4 ) 24
The maximum flexural strain is then derived as More accurate results were further obtained based on the finite-element simulation utilizing the software package COMSOL. The 3D geometry in the model comprised the Ti rod, the steel support blocks, and the cylindrical actuators. The simulated εmax as a function of the load applied is displayed in Fig. S15A, suggesting εmax=0.25% under 2 kN load, which has a better agreement with the 0.21% maximum tensile strain we measured in experiment. The simulated cross-sectional and lateral strain tensor distributions ( Fig. S15B and S15C) quantitatively agree with the experimental strain-mapping results shown in Fig. 4F.

Supplementary Text 4: Strain on the spinal rod measured using the commercial metal-foil strain gauges and off-chip amplifiers.
To further validate the results obtained from the multiplexed single-crystalline silicon piezoresistive sensor arrays and demonstrate their superior performance, we performed the identical four-point bending of the spinal rod with the commercial metal-foil strain gauge (MMF403994, Micro-Measurements) applied on its surface instead (Fig. S8A), as bonded with the same medical-grade polyester tape (3M 1513). The sensor was consisted of a small-section Constantan-alloy filament deposited on a backing plastic substrate. Under applied tensile or compressive strain, the metal wire changes its shape and thus the electrical resistance end-to-end, with a gauge factor of 2. The resistance of the sensor was 120 Ω. During the measurement, it was connected with another three off-chip resistors to form a Wheatstone bridge and the differential voltage signal was amplified with an off-chip amplifier by 1,000×. Since there was no multiplexing capability with the individually packaged commercial sensor, it was sequentially applied to three different locations around the circumference of the spinal rod, as illustrated in Fig.  S8B, to record the strain in three separate load cycles, where the load was increased linearly from 0.1 to 2.1 kN with a rate of 20 N·s -1 . Even after the 1,000× off-chip amplification of the signal and utilizing a much higher drive voltage of 13 V, the data measured using the conventional metalfoil gauge still exhibited much worse signal-to-noise ratio and thus unreliable measurement results compared to what was recorded using the multiplexed single-crystalline silicon piezoresistive sensor arrays, which have ~25 times higher piezoresistive gauge factor and built-in multiplexing for strain mapping, even though the strain measured under the maximum 2 kN load was quantitatively comparable (Fig. S8C).

Supplementary Text 5: Finite-element modeling of the strain distribution on a spinal-rod implant in interbody fusion.
A finite-element model was developed for simulating the biomechanics associated with the posterolateral spinal fusion using COMSOL Multiphysics. The exact geometry of the spinal vertebra in the model was imported from BodyParts3D, a 3D structure database for anatomy (59). As shown in Fig. S9A, the model contained two vertebral bodies with Young's modulus of 12 GPa and an intervertebral spinal disc with a lower modulus of 42.7 MPa (60,61). The backside view of the model shows the facet joints, which were simplified to a cylindrical shape. Their Young's modulus was set to 2.28 MPa for destabilized facet joints (62), and 6 GPa for early-to-middle stage fused facet joints. The spinal rod and screws made of titanium, whose modulus was set as 110 GPa (63), were affixed to the vertebral bodies without loosening. We then simulated the spinal movements in flexion by applying 150 N compression force to the topper part of the vertebral bodies (Fig. S9B). The strain fields in the axial direction of the spinal rods along their surface were estimated and compared between the spinal models with either destabilized or fused facet joints. The simulated strain on the spinal rod demonstrated a large spatial variation, which reflects the importance of straining mapping. The maximum flexural strain reduced significantly after the simulated fusion of facet joints, which agrees both qualitatively and quantitatively with what we observed in experiment.  . Virtually all bacteria were killed by nanopillars, as with flat foils (Fig. 3I). p value for unpaired t-test with unequal variance is 0.002. (E-F) Defect-free aluminum film passivated with four layers of the parylene/Al2O3/HfO2/Al2O3 stacks before (part E) and after (part F) immersion in hot PBS for 30 days. Visible defects start to emerge afterwards. Scale bar: 5 mm.   (7-8-week-old CD-1 mice, 5 per cohort, equal males and females), where the skin barrier was disrupted by the partial removal of the epidermal layer with tape stripping using Tensoplast, followed by the application of the foils inoculated with pathogenic bacteria using surgical tape. (B) Optical images showing the epidermal and subcutaneous tissues, together with the planar controls and the smart-coating foils applied after 3 days, both challenged with 5×10 6 CFUs of P. aeruginosa (278532). Compared to the planar controls, the smart-coating foils with biomimetic mechano-bactericidal nanopillar arrays can effectively prevent the superficial infection as evident from the absence of pus and hemorrhage. (C) Comparison of the S. aureus (N=5) and P. aeruginosa (N=6) burdens in the planar controls (green solid bars), the smart-coating foils featuring antimicrobial nanopillar arrays (green diagonal hatched bars), and their surrounding skin tissues (orange bars). p-values for unpaired t-test between the planar controls and smart-coating foils are all less than 0.0004.